Defining the Translocation Mechanisms of SARS-CoV-2 nsp13 Helicase to Aid in Antiviral Development

定义 SARS-CoV-2 nsp13 解旋酶的易位机制以帮助抗病毒药物开发

基本信息

批准号：
10346024
负责人：
Martin McCullagh
金额：
$ 47.92万
依托单位：
OKLAHOMA STATE UNIVERSITY STILLWATER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-17 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10346024
关键词：
2019-nCoV ATP Hydrolysis ATP phosphohydrolase Address Antiviral Agents Arginine Attenuated Vaccines Behavior Binding Biochemical Biological Assay COVID-19 Catalysis Cessation of life Characteristics Communication Coupled Couples Crystallization Crystallography Data Dengue Development Elements Enzyme Kinetics Enzymes Goals Hydrolysis In Vitro Individual Knowledge Length Ligand Binding Ligands Maps Mechanics Middle East Respiratory Syndrome Modeling Molecular Molecular Conformation Motion Mutagenesis Mutation Nonstructural Protein Nucleotides Pathway Analysis Personal Satisfaction Phenotype Play Process Proteins Protocols documentation RNA RNA Binding RNA Helicase RNA Viruses RNA-Protein Interaction Reaction Research Personnel Resistance SARS coronavirus Site-Directed Mutagenesis Structure Structure-Activity Relationship Subgroup Techniques Temperature Testing Therapeutic Vaccines Viral Viral Proteins Virus Virus Replication Work X-Ray Crystallography analog base combat design enzyme mechanism enzyme structure experience experimental study health economics helicase holistic approach improved in silico inhibitor/antagonist insight molecular dynamics molecular scale multi-scale modeling mutant novel quantum resistance mutation resistant strain response simulation skills targeted treatment therapeutic development tripolyphosphate vaccine development viral RNA

项目摘要

Project Summary SARS-CoV-2, the causative agent of COVID-19, has infected more than 103M people worldwide (February 2021) with more than 2.25M deaths, and represents a dire threat to the health and economic well-being of the entire world. Although vaccines seem to be effective against SARS-CoV-2, recent information regarding potential vaccine resistant strains highlights the importance of alternative strategies to combat this virus. The development of antiviral therapeutics on important mutation resistant viral proteins such as nsp13 is one such strategy. Improved knowledge of the molecular mechanisms utilized by nsp13 are necessary to rationally develop inhibitors. This project will address this deficiency utilizing an integrated multiscale modeling, protein crystallography, and biochemical approach to define how SARS-CoV-2 nsp13 helicase binds RNA and ATP substrates, transduces energy during ATP binding and hydrolysis, and changes conformation during ligand binding and catalysis. We propose the following: 1) Identification of molecular-level components of the RNA- binding and translocation mechanisms of nsp13. Preliminary all-atom molecular dynamics (aaMD) simulations of SARS-CoV-2 nsp13 have identified key protein-RNA interactions that will inform initial mutagenesis studies. Further simulation and protein crystallography will inform on the ATP-dependent protein-RNA interactions observed in the RNA cleft. Biochemical experiments will be performed to test the structure-function hypotheses generated by the structural-based approaches. 2) Identification of molecular-level features of the binding, hydrolysis and product release of ATP by nsp13. We have performed aaMD simulations of the SARS- CoV-2 nsp13 in all relevant substrate states. Soaked-in ATP and non-hydrolysable analogue protein crystallography will be performed to test these initial models. Subsequent quantum mechanical calculations will identify key components of the ATP hydrolysis reaction. Site-directed mutagenesis and well-established enzyme kinetics assays will be used to test effects predicted by these simulations. 3) Identification of allosteric networks in SARS-CoV-2 nsp13 that transduce energy from ATP binding and hydrolysis to perform RNA translocation. Utilizing network analyses of aaMD simulations, Motif V has been identified as a key allosteric contributor. Biochemical studies will be performed to verify that Motif V is necessary for nsp13 helicase function. Further work will be done to identify allosteric networks between additional components of the ATP pocket and RNA cleft identified in Aims 2 and 3. This work will produce unprecedented molecular-level insight into the translocation mechanism of SARS-CoV-2 nsp13 helicases. Key components of this mechanism represent new targets for antiviral development.

项目概要 SARS-CoV-2 是 COVID-19 的病原体，已感染全球超过 1.03 亿人（2 月 2021 年），死亡人数超过 225 万人，对人们的健康和经济福祉构成了严重威胁全世界。尽管疫苗似乎对 SARS-CoV-2 有效，但最近有关的信息潜在的疫苗抗性菌株凸显了对抗这种病毒的替代策略的重要性。这针对重要突变抗性病毒蛋白（例如 nsp13）的抗病毒疗法的开发就是其中之一战略。提高对 nsp13 所利用的分子机制的了解对于合理地开发抑制剂。该项目将利用集成的多尺度建模、蛋白质晶体学和生化方法来定义 SARS-CoV-2 nsp13 解旋酶如何结合 RNA 和 ATP 底物，在 ATP 结合和水解过程中转换能量，并在配体过程中改变构象结合和催化。我们提出以下建议： 1) 鉴定 RNA 的分子水平成分 nsp13 的结合和易位机制。初步全原子分子动力学 (aaMD) 模拟 SARS-CoV-2 nsp13 已确定了关键的蛋白质-RNA 相互作用，这将为初步诱变研究提供信息。进一步的模拟和蛋白质晶体学将揭示 ATP 依赖性蛋白质-RNA 相互作用在 RNA 裂缝中观察到。将进行生化实验来测试结构功能基于结构的方法产生的假设。 2）分子水平特征的鉴定 nsp13 与 ATP 的结合、水解和产物释放。我们对 SARS 进行了 aaMD 模拟所有相关底物状态下的 CoV-2 nsp13。浸泡的 ATP 和不可水解的类似蛋白将进行晶体学测试来测试这些初始模型。随后的量子力学计算将识别 ATP 水解反应的关键成分。定点诱变和成熟酶动力学测定将用于测试这些模拟预测的效果。 3）变构鉴定 SARS-CoV-2 nsp13 中的网络可将 ATP 结合和水解产生的能量转换为 RNA 易位。利用 aaMD 模拟的网络分析，Motif V 已被确定为关键的变构贡献者。将进行生化研究以验证 Motif V 对于 nsp13 解旋酶是必需的功能。将开展进一步的工作来确定 ATP 附加组件之间的变构网络目标 2 和 3 中确定的口袋和 RNA 裂口。这项工作将产生前所未有的分子水平洞察力 SARS-CoV-2 nsp13 解旋酶的易位机制。该机制的关键组成部分代表了抗病毒开发的新目标。